CN105123529A - Rapid propagation and efficient cultivation method of Bletilla striata - Google Patents

Rapid propagation and efficient cultivation method of Bletilla striata Download PDF

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Publication number
CN105123529A
CN105123529A CN201510604885.0A CN201510604885A CN105123529A CN 105123529 A CN105123529 A CN 105123529A CN 201510604885 A CN201510604885 A CN 201510604885A CN 105123529 A CN105123529 A CN 105123529A
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bletilla striata
seedling
medium
tissue culture
light
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CN201510604885.0A
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Inventor
王友海
周洁
李云飞
王昌付
王德春
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YICHANG ACADEMY OF AGRICULTURAL SCIENCE
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YICHANG ACADEMY OF AGRICULTURAL SCIENCE
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Abstract

The invention provides a Bletilla striata tissue cultivation method. The method comprises steps as follows: selected Bletilla striata capsules are washed with water and sterilized; seeds in the Bletilla striata capsules are uniformly shaken off on a first culture medium under the sterile condition and subjected to dark culture for 4.5-5.5 d, and then the seeds are transferred to a culture room for culture under the conditions of predetermined temperature and illumination intensity; Bletilla striata cluster seedlings are inoculated into a second culture medium and cultured under predetermined temperature and illumination intensity until protocorms appear at the base of young seedlings; bottle caps of the young seedlings with the protocorms are opened, the young seedlings are subjected to hardening-seedling for 6-8 days under indoor natural light, and the adaptability of the young seedlings is improved; the young seedlings with the protocorms are taken out, and roots are soaked with carbendazim, and the young seedlings with the protocorms are dried in the air, transplanted to a medium for seedling cultivation and cultured in the shaded, moisturized and ventilated position of a greenhouse at the temperature controlled in a range from 22.5 DEG C to 23.5 DEG C until the young seedlings survive and grow into healthy and strong Bletilla striata plants. With the adoption of the method, fast propagation of Bletilla striata is realized, and the output efficiency of Bletilla striata propagation is improved.

Description

The bletilla striata is numerous and efficient cultivation method soon
Technical field
The present invention relates to plant and cultivate applied technical field, particularly a kind of bletilla striata method for tissue culture.
Background technology
The bletilla striata, formal name used at school Bletillastriata, has another name called Lian Jicao, Gan Gen, gives free of charge, indocalamus is blue, for the orchid family bletilla striata belongs to herbaceos perennial, is mainly distributed in China, Japan and Upper Myanmar.Flower has purplish red, white, blue, yellow and powder isochrome, can potted plant indoor appreciation.There is the effect such as astringing to arrest bleeding, detumescence and promoting granulation, the fields such as extensive use and medicine, daily chemical products, its application constantly expansion in recent years, demand constantly increases, but due to seed development incomplete, be difficult under natural environment sprout, many by stem tuber breeding in production, but reproduction coefficient is limited, has high input, but yield poorly, be difficult to meet the need of market.
Summary of the invention
The invention provides a kind of bletilla striata method for tissue culture, to solve the problem.
Embodiments provide a kind of bletilla striata method for tissue culture, comprise step:
Steps A, carries out sterilization treatment after being rinsed by the bletilla striata capsule water chosen;
Step B, aseptically the seed in described bletilla striata capsule is evenly shaken off on the first medium, carry out light culture 4.5-5.5d, then move to culturing room at predetermined temperature and light according to cultivating, to growing up to the high bletilla striata tufted seedling of 1.8-2.2cm under strength condition;
Step C, is inoculated in the second medium by described bletilla striata tufted seedling, cultivates, until seedling base portion has protocorm to occur according under intensity at predetermined temperature and light;
Step D, opens bottle cap by the seedling of band protocorm, at indoor natural light lower refining seedling 6-8 days, improves seedling adaptability;
Step e, the seedling of the band protocorm obtained in described step D is taken out, clean the medium of root attachment, root 0.5-1h is soaked with 800-1000 times of carbendazim, dry rear transplanting in seedling medium, moisturizing ventilation of sheltering from heat or light in greenhouse is cultivated, and temperature controls at 22.5-23.5 DEG C, until seedling survival, grow up to healthy and strong bletilla striata plant.
Wherein, carry out sterilization treatment in described steps A and comprise step:
Superclean bench adopts 75% ethanol disinfection 28-32s, and 0.1% mercuric chloride sterilization 14-16min, then uses aseptic water washing 3-5 time, suck dry moisture on the filter paper being placed in sterilizing, for subsequent use.
Wherein, described step adopts 75% ethanol disinfection 28-32s on superclean bench, 0.1% mercuric chloride sterilization 14-16min, then comprises for 3-5 time with aseptic water washing:
Superclean bench adopts 75% ethanol disinfection 30s, 0.1% mercuric chloride sterilization 15min, then use aseptic water washing 4 times.
Wherein, described step B comprises step:
Aseptically, cut described bletilla striata capsule top with sterile scalpel, with aseptic nipper, seed is evenly shaken off on the first medium, cover bottle cap immediately, after carrying out mark, be placed on incubator and carry out light culture 5d, then move to culturing room, cultivation temperature 23-27 DEG C, illuminance 2000-2500lx, light irradiation time 11.5-12.5h/d, incubation time is 29-31d, grows up to 2cm height bletilla striata tufted seedling.
Wherein, described step C comprises step:
Be inoculated in the second medium by described bletilla striata tufted seedling, cultivation temperature 23-27 DEG C, illuminance 2000-2500lx, light irradiation time 11.5-12.5h/d, incubation time is 89-91d, to seedling base portion have diameter be 0.5mm protocorm occur.Preferably, being inoculated in the light irradiation time after the second medium in this step is 12h/d, and incubation time is 90d.
Wherein, also step is comprised before described steps A:
Pre-configured first medium, described first medium comprises 1/2MS, 30g/L sucrose, 7g/L agar, 1g/L active carbon, and ph is 5.8.
Wherein, also step is comprised before described steps A:
Pre-configured second medium, described second medium comprises MS, 0.75mg/LNAA, 0.5mg/L6-BA, 25g/L sucrose, 200g/L potato, 6.5g/L agar, 5g/L active carbon, and ph is 5.8.
Wherein, step is comprised before described step e:
Configuration seedling medium, described seedling medium comprises humus soil, perlite and river sand.
Wherein, the composition in described seedling medium by volume number counts humus soil: perlite: river sand=2: 1: 1.
Embodiments provide a kind of bletilla striata method for tissue culture, with the excellent bletilla striata seeds of proterties for explant, disinfection under specific temperature light degree condition, inoculation induction is sprouted, squamous subculture, grow up to complete bletilla striata plant seedling, finally by complete little transplantation of seedlings in seedling medium, be trained bletilla striata seedling, whole incubation standard is controlled, achieve the batch production standard production of bletilla striata seedling, reproduction speed is fast, all can breed production seedling throughout the year, not by region and climatic effect, compare traditional wild excavating to cultivate with artificial stem tuber, production process is controlled, the rate of output is high, output is larger, solve the bletilla striata in prior art and cultivate the low technical problem being difficult to meet market demand of output capacity.
Accompanying drawing explanation
Fig. 1 is the flow chart of an embodiment of bletilla striata method for tissue culture provided by the invention.
Embodiment
Embodiments provide a kind of bletilla striata method for tissue culture.Shown in Figure 1, the method comprising the steps of:
Step S110, carries out sterilization treatment after being rinsed by the bletilla striata capsule water chosen.
The ripe capsule of the bletilla striata plant that bletilla striata capsule should select shape excellent.
Preferably, sterilization treatment in the following way: on superclean bench, adopt 75% ethanol disinfection 28-32s, 0.1% mercuric chloride sterilization 14-16min, then uses aseptic water washing 3-5 time, suck dry moisture on the filter paper being placed in sterilizing, for subsequent use.
Wherein, in the present invention unless stated otherwise, s is time measurement unit second, min is time measurement unit minute, and d is time measurement unit sky, and h is time measurement unit hour, mm is length metering unit millimeter, and cm is length metering unit centimetre, and lx is intensity of illumination unit lux.
Step S111, aseptically the seed in described bletilla striata capsule is evenly shaken off on the first medium, carry out light culture 4.5-5.5d, then move to culturing room at predetermined temperature and light according to cultivating, to growing up to the high bletilla striata tufted seedling of 1.8-2.2cm under strength condition.
Step S112, is inoculated in bletilla striata tufted seedling in the second medium, cultivates, until seedling base portion has protocorm to occur according under intensity at predetermined temperature and light.
In this step, predetermined temperature and light according to intensity can be: cultivation temperature 23-27 DEG C, illuminance 2000-2500lx; And light irradiation time can be 11.5-12.5h/d, incubation time is 29-31d.
Step S113, opens bottle cap by the seedling of band protocorm, at indoor natural light lower refining seedling 6-8 days, improves seedling adaptability.
Step S114, the seedling of the band protocorm obtained in described step S113 is taken out, clean the medium of root attachment, root 0.5-1h is soaked with 800-1000 times of carbendazim, dry rear transplanting in seedling medium, moisturizing ventilation of sheltering from heat or light in greenhouse is cultivated, and temperature controls at 22.5-23.5 DEG C, until seedling survival, grow up to healthy and strong bletilla striata plant.
Seedling medium, by volume number meter are humus soil: perlite: sand=2, river: 1: 1.
Wherein, the first medium is seed germination medium, and the second medium is subculture medium.Preferably, as a kind of embodiment, the first medium comprises: 1/2MS+30g/L sucrose+7g/L agar+1g/L active carbon, the first medium ph value is 5.8.
Second medium comprises: MS+0.75mg/LNAA+0.5mg/L6-BA+25g/L sucrose+200g/L potato+6.5g/L agar+5g/L active carbon, the second medium ph value is 5.8.
Wherein, MS is conventional minimal medium Murashige & Skoog1962, and namely Murashige and Sk0og was the conventional medium of tobacco cell Training Design in 1962.Macroelement concentration in conventional minimal medium MS is reduced by half and namely obtains 1/2MS; 6-BA is 6-benzyladenine, and NAA is methyl α-naphthyl acetate.
Enumerate a preferred embodiment of a bletilla striata method for tissue culture provided by the invention below, its step is as follows:
1) the choosing and sterilizing of bletilla striata seeds capsule: choose ripe bletilla striata capsule, bletilla striata capsule water is rinsed well, superclean bench adopts 75% ethanol disinfection 30s, 0.1% mercuric chloride sterilization 15min, use aseptic water washing again 4 times, suck dry moisture on the filter paper being placed in sterilizing, for subsequent use.
2) inoculate: aseptically, cut capsule top with sterile scalpel, with aseptic nipper, seed is evenly shaken off on medium, cover bottle cap immediately, after carrying out mark, be placed on incubator and carry out light culture 5d, then move to culturing room, cultivation temperature 25 ± 2 DEG C, illuminance 2000-2500lx, illumination 12h/d, incubation time is 30d, grows up to 2cm height bletilla striata tufted seedling.
3) squamous subculture: the bletilla striata tufted seedling in step 2 is inoculated in subculture medium, cultivation temperature (25 ± 2) DEG C, illuminance 2000 ~ 2500lx, illumination 12h/d, incubation time is 90d, have diameter be 0.5mm (millimeter) protocorm occur, seedling base portion slightly expands.
4) plant hardening: the seedling of the band protocorm obtained in step 3 is opened bottle cap, indoor natural light lower refining seedling about a week, improves seedling adaptability.
5) plantlet of transplant: before transplanting, carry out disinfection to containing humus soil, perlitic seedling medium, the seedling of the band protocorm obtained in step 4 is taken out, cleans base portion medium, soak root 0.5-1h with 800-1000 times of carbendazim, dry rear transplanting in seedling medium, moisturizing ventilation of sheltering from heat or light in greenhouse is cultivated, and temperature controls at about 23 DEG C, and matrix keeps " see dry see wet ", until seedling survival, grow up to healthy and strong bletilla striata plant.
After cultivating into healthy and strong bletilla striata plant, carry out field production and comprise step: by transplanting survival, the bletilla striata seedling of robust growth transplants in spring and autumn, select that Schattenseite sparse woods place, the soil organic matter are abundant, well-drained sandy loam or loam plant.Preferably, can furrow height 25cm, wide 1.2m, seeding row spacing 15 × 25cm, according to the annual weeding of bletilla striata growing state 3 times, be respectively the 3-4 month, June, the 8-9 month, shallow except table soil during weeding, do not hinder stem tuber.Select mountain forest hillside fields plantation bletilla striata damage by disease and insect relatively less, mainly carry out drainage works, prevent underground stem tuber from rotting.Be rich in the forest land of humus substantially without artificially applying fertilizer, if land fertility is not enough, potassium dihydrogen phosphate or compoiste fertilizer can be imposed in conjunction with intertill and clean tillage.Plantlet in vitro is excavated after generally growing 3 years, underwood planting per mu yield 250kg.
Select the Schattenseite forest land plantation bletilla striata, both can improve land utilization rate, and in turn simplify the Cultivate administration of the bletilla striata, and reduce human cost, increase economic efficiency.
In prior art; the bletilla striata mostly is wild and excavates and artificial cultivation; cultivation is based on stem tuber breeding; plant development cost is high, and stem tuber easily rots in soil, and disease resistance insufficient strength; plant for many years and easily occur deterioration of variety; provenance source is numerous and diverse simultaneously, and quality is uneven, constrains the development of the large-scale planting of the bletilla striata and the raising of the unit are output value.The correlation technique that there is no at present about bletilla striata tissue cultures and rapid propagation in vitro solves the low problem of bletilla striata stem tuber reproduction coefficient in production.
Bletilla striata method for tissue culture provided by the invention, utilize plant tissue culture technique, realize the tissue rapid propagation of the bletilla striata, reproduction coefficient is high, for later factorial seedling growth provides certain technical support, in the seedling supply of cultivating in the industrialization of the bletilla striata and breeding fast-propagation, there is important using value:
1, realize the batch production standard production of bletilla striata seedling, reproduction speed is fast, all can breed production seedling throughout the year, not by region and climatic effect;
2, fine-variety breeding is numerous with expansion fast, carries out fast-propagation, and realize the quick breeding of breeding on this basis for elite plant, for large-scale artificial cultivation provides good seed;
3, for bletilla striata Germ-plasma resources protection provides low-cost technologies means.
It should be noted that, in this article, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, thus make to comprise the process of a series of key element, method, article or equipment and not only comprise those key elements, but also comprise other key elements clearly do not listed, or also comprise by the intrinsic key element of this process, method, article person equipment.When not more restrictions, the key element " being comprised " limited by statement, and be not precluded within process, method, article or the equipment comprising described key element and also there is other identical factor.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment made, equivalent replacement, improvement etc., all should be included within the scope of protection of the invention.

Claims (9)

1. a bletilla striata method for tissue culture, is characterized in that, comprises step:
Steps A, carries out sterilization treatment after being rinsed by the bletilla striata capsule water chosen;
Step B, aseptically the seed in described bletilla striata capsule is evenly shaken off on the first medium, carry out light culture 4.5-5.5d, then move to culturing room at predetermined temperature and light according to cultivating, to growing up to the high bletilla striata tufted seedling of 1.8-2.2cm under strength condition;
Step C, is inoculated in the second medium by described bletilla striata tufted seedling, cultivates, until seedling base portion has protocorm to occur according under intensity at predetermined temperature and light;
Step D, opens bottle cap by the seedling of band protocorm, at indoor natural light lower refining seedling 6-8 days, improves seedling adaptability;
Step e, the seedling of the band protocorm obtained in described step D is taken out, clean the medium of root attachment, root 0.5-1h is soaked with 800-1000 times of carbendazim, dry rear transplanting in seedling medium, moisturizing ventilation of sheltering from heat or light in greenhouse is cultivated, and temperature controls at 22.5-23.5 DEG C, until seedling survival, grow up to healthy and strong bletilla striata plant.
2. bletilla striata method for tissue culture according to claim 1, is characterized in that, carries out sterilization treatment and comprise step in described steps A:
Superclean bench adopts 75% ethanol disinfection 28-32s, and 0.1% mercuric chloride sterilization 14-16min, then uses aseptic water washing 3-5 time, suck dry moisture on the filter paper being placed in sterilizing, for subsequent use.
3. bletilla striata method for tissue culture according to claim 2, is characterized in that, described step adopts 75% ethanol disinfection 28-32s on superclean bench, 0.1% mercuric chloride sterilization 14-16min, then comprises for 3-5 time with aseptic water washing:
Superclean bench adopts 75% ethanol disinfection 30s, 0.1% mercuric chloride sterilization 15min, then use aseptic water washing 4 times.
4. bletilla striata method for tissue culture according to claim 1, is characterized in that, described step B comprises step:
Aseptically, cut described bletilla striata capsule top with sterile scalpel, with aseptic nipper, seed is evenly shaken off on the first medium, cover bottle cap immediately, after carrying out mark, be placed on incubator and carry out light culture 5d, then move to culturing room, cultivation temperature 23-27 DEG C, illuminance 2000-2500lx, light irradiation time 11.5-12.5h/d, incubation time is 29-31d, grows up to 2cm height bletilla striata tufted seedling.
5. bletilla striata method for tissue culture according to claim 1, is characterized in that, described step C comprises step:
Be inoculated in the second medium by described bletilla striata tufted seedling, cultivation temperature 23-27 DEG C, illuminance 2000-2500lx, light irradiation time 11.5-12.5h/d, incubation time is 89-91d, to seedling base portion have diameter be 0.5mm protocorm occur.
6. bletilla striata method for tissue culture according to claim 1, is characterized in that, also comprises step before described steps A:
Pre-configured first medium, described first medium comprises 1/2MS, 30g/L sucrose, 7g/L agar, 1g/L active carbon, and ph value is 5.8.
7. bletilla striata method for tissue culture according to claim 1, is characterized in that, also comprises step before described steps A:
Pre-configured second medium, described second medium comprises MS, 0.75mg/LNAA, 0.5mg/L6-BA, 25g/L sucrose, 200g/L potato, 6.5g/L agar, 5g/L active carbon, and ph value is 5.8.
8. bletilla striata method for tissue culture according to claim 1, is characterized in that, comprises step before described step e:
Configuration seedling medium, described seedling medium comprises humus soil, perlite and river sand.
9. bletilla striata method for tissue culture according to claim 8, is characterized in that, the composition in described seedling medium by volume number counts humus soil: perlite: sand=2, river: 1: 1.
CN201510604885.0A 2015-09-22 2015-09-22 Rapid propagation and efficient cultivation method of Bletilla striata Pending CN105123529A (en)

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CN105830582A (en) * 2016-04-15 2016-08-10 成都大学 Quickbackfillcultivation method of rhizoma bletillae seeds
CN105993278A (en) * 2016-05-18 2016-10-12 四川新绿色药业科技发展股份有限公司 Convenient and low-cost method for promoting emergence of bletilla striata seeds
CN106068790A (en) * 2016-07-19 2016-11-09 江苏省中国科学院植物研究所 A kind of method of bletilla striata seeds direct sowing and seedling
CN106417027A (en) * 2016-10-14 2017-02-22 贵州万重山生态农业开发有限公司 Low-temperature seedling hardening method for bletilla striata tissue culture seedlings
CN107047310A (en) * 2017-05-10 2017-08-18 文山学院 A kind of cultural method of bletilla striata seeds culture seedling
CN109169275A (en) * 2018-09-13 2019-01-11 甘肃源宜生物科技有限公司 A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method
CN109964815A (en) * 2019-03-26 2019-07-05 成都大学 A kind of bletilla striata aseptic seedling rapid induction Multiple Buds and fast numerous method of taking root

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105830582A (en) * 2016-04-15 2016-08-10 成都大学 Quickbackfillcultivation method of rhizoma bletillae seeds
CN105830582B (en) * 2016-04-15 2018-05-01 成都大学 A kind of the quick of bletilla seed returns native breeding method
CN105993278A (en) * 2016-05-18 2016-10-12 四川新绿色药业科技发展股份有限公司 Convenient and low-cost method for promoting emergence of bletilla striata seeds
CN105993278B (en) * 2016-05-18 2021-11-23 四川新绿色药业科技发展有限公司 Convenient and low-cost method for promoting bletilla striata seed emergence
CN106068790A (en) * 2016-07-19 2016-11-09 江苏省中国科学院植物研究所 A kind of method of bletilla striata seeds direct sowing and seedling
CN106068790B (en) * 2016-07-19 2018-11-16 江苏省中国科学院植物研究所 A kind of method of bletilla striata seeds direct sowing and seedling
CN106417027A (en) * 2016-10-14 2017-02-22 贵州万重山生态农业开发有限公司 Low-temperature seedling hardening method for bletilla striata tissue culture seedlings
CN107047310A (en) * 2017-05-10 2017-08-18 文山学院 A kind of cultural method of bletilla striata seeds culture seedling
CN109169275A (en) * 2018-09-13 2019-01-11 甘肃源宜生物科技有限公司 A kind of pale reddish brown trident bletilla striata tissue-culturing rapid propagation culture medium and method
CN109964815A (en) * 2019-03-26 2019-07-05 成都大学 A kind of bletilla striata aseptic seedling rapid induction Multiple Buds and fast numerous method of taking root

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